Many bacteria, including Agrobacterium tumefaciens, rely on an asymmetric localization, distribution, and orientation of specific appendages when interacting with potential hosts and with their environment. The study of developmental asymmetry is well advanced in the model a-Proteobacterial organism Caulobacter crescentus where the interlinked PleC/DivJ-DivK and CckA-ChpT-CtrA/CpdR His-Asp phosphorelays play a central role. Despite a high degree of conservation among the a-Proteobacteria, including A. tumefaciens little is known about the precise role of these regulatory proteins in these other organisms. Two related proteins, the PleC-DivJ homologue sensor kinases PdhS1 and PdhS2, are restricted to a subset of a-Proteobacteria that frequently associate with a eukaryotic host in a symbiotic or pathogenic relationship. This study examines the role of these regulatory proteins in A. tumefaciens. In particular, this application addresses how PdhS1 and PdhS2 contribute to the morphological, physiological, and temporal development of A. tumefaciens and whether these processes contribute to the transition between host and non-host niches. This will be performed using a combination of genetic, biochemical, and cytological analyses. PdhS1 and PdhS2 will both be biochemically dissected to determine the contribution of individual catalytic and regulatory domains to their activity and to identify potential interacting partners. A. tumefaciens binds to abiotic and biotic surfaces in a polar orientation and is known to elaborate polar flagella, pili, and a type IV secretion system. In addition, A. tumefaciens and many other a-Proteobacteria divide by an asymmetric process of budding, proceeding through an attached, non-motile form to a morphologically distinct motile form. Phenotypic analysis will evaluate the contribution of PdhS1 and PdhS2 to polar development, motility, biofilm formation, and virulence. Additional genes and regulatory interactions that influence coordination of polar morphogenesis, motility, and biofilm formation in A. tumefaciens will be identified using a combination of transposon mutagenesis and a novel high-throughput microscopy screen. PUBLIC HEALTH RELEVANCE: I am using the bacterial pathogen Agrobacterium tumefaciens as a model organism for studying how bacteria regulate their multiplication and shape, and how this impacts their interactions with host organisms. In particular, I am studying two key regulatory proteins that allow A. tumefaciens to integrate environmental signals and transition from the free-living to the host-associated state. These proteins are conserved among a range of pathogenic and non-pathogenic host-associated bacteria and therefore any insights gained from this project will be more broadly applicable to other disease agents.